An Academic Research Enhancement Award (AREA) project proposes to identify small RNA molecules and to determine the role of the global regulator Hfq in the biothreat agent, Francisella tularensis. This bacterium is considered a category A agent due to its high infectivity and virulence (manifested by the fact that as few as ten organisms can cause potentially fatal disease in humans), documented production for potential deployment in biological warfare, ease of microbial culture, and genetic tractability. The long-term goal of this project is to better understand the role of Hfq and small RNAs in post-transcriptional regulation in this important pathogen. The immediate focus of this pilot study is to determine the role of Hfq in adaptive responses to stress and potential contribution towards the ability of Francisella to survive and replicate in macrophages (an essential virulence attribute). The identification of small RNAs is a critical adjunct to Hfq regulation as a subset of such transcripts may collaborate with Hfq to assert regulatory effects. The presence of small RNAs and the burgeoning field of RNomics, has not been addressed in this organism. Importantly, in silico identification is compounded by the absence of closely related genome sequences in which small RNAs have been identified. Accordingly, molecular and biochemical strategies are proposed to identify small RNAs, which are anticipated to have novel sequences and affect diverse functions, including roles in Francisella pathobiology. The Specific Aims integrate genomic, molecular, biochemical, and cellular microbiological approaches to: i) determine the role of Hfq in stress responses and in intra-macrophage survival and replication, ii) identify small non-coding RNAs in Francisella tularensis, and iii) assess expression features of candidate sRNAs. This proposal addresses a novel aspect of the basic biology of a threat agent and will provide new information and insight into post-transcriptional regulatory systems that are being increasingly recognized as important in orchestrating regulation of "virulome" components in multiple pathogens, but that are unexplored in Francisella. Furthermore, if Hfq or small RNAs are implicated in the regulation of virulence gene expression, the data generated by completion of the Specific Aims may provide pivotal insight for rational vaccine design and should establish a platform from which strategies for immune or chemotherapeutic intervention can be devised and implemented. PUBLIC HEALTH RELEVANCE: The proposed research is expected to identify novel regulatory molecules that are critical to the survival and pathogenesis of the biothreat agent Francisella tularensis. The study of these regulatory targets will provide unprecedented insight for the future development of strategies for treatment and prevention of the potentially fatal disease, tularemia.